TAB1 protein and DNA coding therefore

ABSTRACT

TAB1 protein having activity which activates factor TAK1 in the TGF-β signaling pathway, and having the amino acid sequence shown in FIG.  1.

BACKGROUND OF INVENTION

1. Field of Invention

The present invention relates to TAB1 protein which forms a part of thesignal-transduction pathway of Transforming Growth Factor-β (TGFβ), andto a gene coding therefor.

2. Related Art

TGF-β is a multifunctional factor which regulates various cellularfunctions. As one of those functions, TGF-β is responsible for therepair and reproduction of tissues with various types of injury.

Abnormal production of TGF-β in cases of chronic injury sometimesresults in an imbalance between the repair and the reproduction oftissues and thus pathological fibrosis. Hepatic fibrosis is known as onecondition resulting from an imbalance in TGF-β production. In the liver,TGF-β accelerates production of extracellular matrix proteins which areresponsible for fibrosis, while inhibiting synthesis of extracellularmatrix protein catabolic enzymes and inducing catabolic enzymeinhibitors, and it thus acts as a major causative factor of hepaticfibrosis.

One of known members of signal-transduction pathway belonging to theTGF-β superfamily is the Mitogen-Activated Protein Kinase Kinase Kinase(MAPKKK) system.

The MAPK pathway is a conserved eukaryotic signal-transduction pathwaywhich converts receptor signals into various functions, and the systemcomprises 3 different protein kinases, specifically MAPKKK mentionedabove, MAPKK and MAPK, with MAPK being activated through phosphorylationby MAPKK, and MAPKK in turn being activated by MAPKKK (E. Nishida etal., Trends Biochem. Sci. Vol. 18, p. 128 (1993); K. J. Blumer et al,op. cit. Vol. 19, p. 236 (1994); R. J. David, op. cit. Vol. 19, p. 470(1994); C. J. Marchall, Cell, Vol. 80, p. 179 (1995)).

TAK1, which is a member of the MAPKKK family which functions insignal-transduction pathways belonging to the TGF-β superfamily, hasbeen identified by K. Yamaguchi (K. Yamaguchi et al., Science, Vol. 270,p. 2008 (1995)).

TGF-β transduces signal through a heteromeric complex of type I and typeII TGF-β receptors, which are transmembrane proteins comprisingcytoplasmic serine- and threonine-specific kinase domains (J. L. Wranaet al., Nature, Vol. 370, p. 341 (1994); D. M. Kingsley et al.,. GenesDev., Vol. 8, p. 133 (1994)). However, little is known at the molecularlevel about the signal-transduction mechanism downstream from the TGF-βreceptors.

SUMMARY OF INVENTION

It is an object of the present invention, therefore, to provide TAB1protein which is a newly discovered member in the TGF-β receptorsignal-transduction pathway, and to a gene coding therefor. The presentinvention further provides a screening method for TGF-βsignal-transduction pathway inhibitors. TAB1 refers to a protein whichbinds to TAK1 (TAK1 Binding protein).

In order to achieve these objects, the present invention provides TAB1protein having the amino acid sequence shown in SEQ ID NO: 1; a proteinhaving an amino acid sequence shown in SEQ ID NO: 1 modified bysubstitution, deletion and/or addition of one or more amino acids in theamino acid sequence shown in SEQ ID NO: 1, and having a biologicalproperty of TAB1 protein; a protein wherein the 52nd amino acid of theamino acid sequence shown in SEQ ID NO: 1 is arginine; a protein encodedby DNA which can hybridize with DNA having the nucleotide sequence shownin SEQ ID NO: 7 under hybridization conditions of 60° C., 0.1×SSC, 0.1%sodium dodecyl sulfate, and which has a biological property of TAB1protein; a protein having an amino acid sequence consisting of aminoacids from amino acid positions 21 to 579 of the amino acid sequenceshown in SEQ ID NO: 1; and a polypeptide having the amino acid sequenceconsisting of the 68 amino acids from amino acid positions 437 to 504 ofthe amino acid sequence shown in SEQ ID NO: 1.

The present invention further provides a method for producing any of theabove-mentioned proteins or polypeptides comprising the steps ofculturing a host transformed by an expression vector comprising DNAencoding the protein or polypeptide, and recovering the protein orpolypeptide from the culture.

The present invention still further provides a method for inducingmammalian cells to produce any of the above-mentioned proteins orpolypeptides comprising the step of introducing DNA encoding the proteinor polypeptide into mammalian cells.

The present invention still further provides DNA encoding any of theabove-mentioned proteins or polypeptides, an expression vectorcomprising the DNA, and a host transformed by the expression vector.

The present invention still further provides a method for screeningTGF-β signal-transduction pathway inhibitors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the regions on the TAK1 protein to which TAB1 proteinbinds. The shaded areas indicate the TAK1 catalytic domain.

FIG. 2 shows complementation of the Stell deletion by the copresence ofTAK1 and TAB1 in an Ste11 deletion strain, in the pheromone-activatedMAPK pathway of yeast.

FIG. 3 is the results of electrophoresis shown in a photographs showingthe results of the in vitro experiment indicating reinforcement of TAK1activity by TAB1.

FIG. 4 shows the amino acid sequence of TAB1 (SEQ ID NO: 1) with aninsertion of partial TAK1 sequence (SEQ ID NO: 8) for comparison.

FIG. 5 is an electrophoresis diagram showing expression of mRNA codingfor TAB1 in various organs and tissues.

FIG. 6 is an immunoblot diagram showing association of TAB1 and TAK1 inmammalian cells.

FIG. 7 contains a graph showing enhancement of TAK1 kinase activity byTAB1 in mammalian cells (top), and a blot diagram showing comparableamounts of production of TAK1 and KN-MPK2.

FIG. 8 is a graph showing enhanced expression of a luciferase reportergene by the copresence of TAK1 and TAB1 in mammalian cells stimulated byTGF-β.

FIG. 9 is a graph showing inhibition of the TGF-β-induced luciferasereporter gene expression by TAB-lacking the C-terminus (TAB1 (1-418)).

DETAILED DESCRIPTION

The TAB1 protein according to the invention has the characteristic ofactivating TAK1 by binding TAK1 in the signal-transduction pathway oftransforming growth factor-β(TGF-β). This and other characteristics aredescribed in detail Examples 2 to 4 and 6 to 10.

The TAB1 protein of the invention has the amino acid sequence (SEQ IDNO: 1) derived from the nucleotide sequence of cDNA cloned by the methoddescribed in Examples 1 and 5. However, it is well known that proteinswith biological activity exist whose amino acid sequences have beenmodified by a substitution, deletion and/or addition of one or moreamino acids, and which maintain a biological property of the wildprotein. Thus, the present invention encompasses proteins having anamino acid sequence modified by a substitution, deletion and/or additionof one or more amino acids in the amino acid sequence shown in SEQ IDNO: 1, and having a biological property of TAB1 protein.

One embodiment thereof is a protein wherein the 52nd amino acid of theamino acid sequence shown in SEQ ID NO: 1 is arginine.

It is also known that once DNA coding for a specific protein has beencloned, the DNA may be used as a probe for screening of a DNA libraryfrom organs or tissue different from the organs or tissue from which theprotein was obtained, or a DNA library from another species, to obtainDNA coding for a protein with similar biological property though havinga different amino acid sequence. Thus, the present invention alsoencompasses proteins encoded by DNA which can hybridize with DNA havingthe nucleotide sequence shown in SEQ ID NO: 7 under hybridizationconditions of 60° C., 0.1×SSC, 0.1% sodium dodecyl sulfate, and whichhas a biological property of TAB1 protein.

An example of a modified protein according to the invention is a proteinhaving an amino acid sequence consisting of amino acids from amino acidpositions 21 to 579 of the amino acid sequence shown in SEQ ID NO: 1.This protein has the biological property of TAB1 protein. An instance ofa modified polypeptide according to the invention is a polypeptidehaving the amino acid sequence consisting of the 68 amino acids fromamino acid positions 437 to 504 of the amino acid sequence shown in SEQID NO: 1. This polypeptide has the properties of activating TAK1 kinaseactivity upon binding to TAK1.

Another example of a modified protein according to the invention is afused protein between the aforementioned protein or polypeptide andanother protein, which has a biological activity of TAB1.

Proteins or polypeptides of the invention can imitate the actualphysiological function of TGF-β by, for example, activating TAK1 whichis important to the TGF-β signal-transduction pathway, as well asinhibit binding between TAK1 and TAB1 by their binding to TAK1, and theyare therefore useful for methods of screening substances which act asagonists or antagonists against cell growth suppression,immunosuppression and bone differentiation.

DNA coding for a protein of the invention is, for example, DNA codingfor the amino acid sequence shown in SEQ ID NO: 1. Such DNA may beobtained, for example, by the method described in Examples 1 and 5, andit has the nucleotide sequence shown in SEQ ID NO: 7. However, DNAcoding for the amino acid sequence shown in SEQ ID NO: 1 does notnecessarily have the nucleotide sequence shown in SEQ ID NO: 7, as itmay consist of other codons coding for the same amino acids. Forexample, the human derived nucleotide sequence shown in SEQ ID NO: 7 maybe altered to include a codon which is efficiently translated in suchmicroorganisms as bacteria or yeast, and this may be accomplished usinga well-known technique such as site-specific mutagenesis with a primer.

DNA according to the invention coding for a protein or polypeptidehaving an amino acid sequence modified by a substitution, deletionand/or addition of one or more amino acids in the amino acid sequenceshown in SEQ ID NO: 1 may be prepared by a well-known method such assite-specific mutagenesis or the PCR, using DNA with the nucleotidesequence shown in SEQ ID NO: 7 as the template. Alternatively, DNAcoding for a protein or polypeptide wherein the modified amino acidsequence is shorter than the natural protein may be obtained, forexample, by introducing a translation initiation codon and/ortranslation termination codon into naturally occurring DNA, such ascDNA. The introduction of these codons may be accomplished bysite-specific mutagenesis or the PCR. Alternatively, it may be achievedby cleaving the natural DNA, such as cDNA, with an appropriaterestriction enzyme, and adding the desired oligonucleotide if necessary.

DNA which can be hybridized with DNA having the nucleotide sequenceshown in SEQ ID NO: 7 of the invention and which codes for a proteinhaving a biological property of TAB1 may be obtained by screening agenomic DNA library or cDNA library prepared, for example, from thevarious tissues and organs mentioned in Example 6, including the heart,brain, placenta, liver, skeletal muscle, kidney, pancreas, spleen,thymus, prostate, testicles, ovaries, small intestine, colon, peripheralleukocytes, etc., using the nucleotide sequence shown in SEQ ID NO: 7 ofthe invention or a portion thereof as the probe. The DNA library is notlimited to a human derived one, and may be derived from other animalssuch as rats, mice, rabbits, goats, sheep, cattle or pigs.

The present invention also relates to an expression vector comprising anaforementioned DNA and to a host transformed therewith. The expressionvector will differ depending on the host. The host cells used accordingto the invention may be from any prokaryotic or eukaryotic organisms.The prokaryotic organisms used may be bacteria, for example,microorganisms belonging to the genus Escherichia such as Escherichiacoli, microorganisms belonging to the genus Bacillus such as Bacillussubtilis, etc., and the eukaryotic organisms may be lower eukaryoticorganisms, such as filamentous fungi and yeast.

Filamentous fungi include microorganisms belonging to the genusAspergillus such as Aspergillus niger and Aspergillus orizae andmicroorganisms belonging to the genus Penicillium, while the yeast maybe microorganisms belonging to the genus Saccharomyces such asSaccharomyces cerevisiae.

Higher eukaryotic organisms which may be used include various animal andplant cells, for example, immortralized cultured animal cells such asCOS cells, CHO cells and NIH3T3, etc. Insect cells such as Sf9, Sf12,etc. may also be used.

The expression vector of the invention includes, in addition to DNAcoding for a protein or polypeptide of the invention, expressionregulating sequences, such as promoters, which are functionable in thehost.

Promoters for bacteria, for example E. coli, include T3 and T7, whilepromoters for yeast include glycolytic enzyme gene promoters such asGAL1 promoter and GAL4 promoter. The promoter for animal cells may be aviral promoter, such as CMV promoter or SV40 promoter.

The transformation of the host by an expression vector, culturing thehost, and the collection and purification of the protein or polypeptideof the invention from the culture may be accomplished according toconventional methods. For example, the isolation and purification of theprotein or polypeptide from the culture may be accomplished using anyconventional means for isolating and purifying proteins andpolypeptides, such as ammonium sulfate precipitation, gel filtration orreverse phase HPLC, either alone or in combinations.

The present invention also relates to a screening method for TGF-βsignal-transduction pathway inhibitors. A sample containing TGF-βsignal-transduction pathway inhibitors is brought to contact with orintroduced into cells expressing a protein with a biological activityTAB1 and TAK1 (K. Yamaguchi et al., Science, Vol. 270, p. 2008 (1995)),and the TAK1 activity is then measured. The protein or polypeptide withbiological activity of TAB1 and TAK1 may also be fused with anotherprotein, and the cells expressing them may be yeast cells or mammaliancells. This screening system may be constructed according to the methoddescribed in Examples 1, 2, 3, 4, 7, 8 and 9.

The sample containing TGF-β signal-transduction pathway inhibitors isbrought to contact with or introduced into the constructed screeningsystem, and the TAK1 kinase activity is measured. The method formeasuring the TAK1 kinase activity may be measurement of the kinaseactivity of TAK1 itself, or measurement of the kinase activity of MAPKKor MAPK which are downstream from TAK1 in the signal-transductionpathway and are activated by TAK1. The activity of a target gene in theMAPK pathway or a reporter gene under the control of the target genepromoter may also be measured based on the amount of mRNA or expressedform of the gene.

The screening method for TGF-β signal-transduction pathway inhibitorsaccording to the invention allows screening of substances which inhibitbinding between TAB1 and TAK1 and can thus serve as a means of therapyfor diseases involving abnormal-production of TGF-β.

EXAMPLES

The present invention will now be explained in more detail by way of thefollowing examples.

Example 1

Analysis of the TAK1-dependent pathway functioning for TGF-βsignal-transduction was made using a yeast 2-hybrid system (S. Frelds etal., Trend Genet. 10, 286 (1994)), and a protein having directinteraction with TAK1 was sought.

First, an expression vector was constructed by linking the TAK1 gene anda gene coding for the LexA DNA-binding domain. pLexA-TAK1Δ contains theTAK1ΔN coding sequence (K. Yamaguchi et-al., Science, Vol. 270, p. 2008(1995)) inserted in frame into pBTM116 (A. B. Vojtek et al., Cell, Vol.74, p. 205 (1993)). A yeast 2-hybrid system was used to identify aprotein encoded in a human brain cDNA library and interacting withTAK1ΔN.

The two hybrids were expressed in Saccharomyces cerevisiae L40(LYS2:LexA-HIS3) containing an integrated reporter construct with abinding site for LexA protein located upstream from the yeast HIS3coding region. Interaction between the two hybrid proteins causestransactivation of the reporter construct, allowing growth of the yeastin the absence of histidine (SC-His).

The LexA-TAK1ΔN fused protein alone confers expression of HIS3 in asufficient amount to allow growth without requiring exogenous histidine.However, histidine auxotrophy can be achieved by growing the cells inthe presence of 40 mm 3-aminotriazole (3-AT) which is a chemicalinhibitor of the HIS3 gene product imidazole glycerol dehydrogenase (G.M. Kishore et al., Annu. Rev. Biochem. Vol. 57, p. 627 (1988)).

Yeast was transformed using a bait plasmid together with a fish plasmidcontaining the human brain cDNA expression library clone linked to thegene coding for the GAL4 activating domain (GAD). A positive clone ofTAB1 cDNA coding for the protein was obtained from about 1×10⁶transformants. The GAD fused protein expressed by this isolated DNA willhereinafter be referred to as GAD-TAB 1.

Example 2

A series of LexA-TAK1 deletion chimera were tested by the 2-hybridmethod to determine the site in TAK1 which is responsible forinteraction with TAB1. An expression vector coding for the full TAK-1 ordeletion construct thereof fused to the LexA DNA-binding domain was usedfor simultaneous transformation of the yeast reporter strain L40together with pGAD-TAB1. The DNA coding for each of the TAK1 deletionconstructs was prepared from DNA coding for the full TAK1.

The aforementioned plasmid pGAD-TAB1 was obtained by cloning TAB1 cDNAat the EcoRI site of pBS (W. O. Bullock et al., Biotechniques, Vol. 5,p. 376 (1987)). The interaction between the fused proteins expressed bythis plasmid is indicated by the ability of the yeast strain, to grow ona plate of SC—HIS medium containing 40 mM 3-AT. The results are shown inFIG. 1. The right side of this graph indicates whether TAK1 or itsdeletion form interacted with TAB1 (+) or not (−). These resultsdemonstrate that TAB1 interacts with the N-terminal domain of TAK1.

Example 3

A protein interacting with TAK1 may contain both the upstream controlregion and the downstream target. If TAB1 plays a role in activation ofTAK1, then their simultaneous expression would be expected to influenceactivity of TAK1 in yeast. The present inventors have disclosed a systemfor assaying mammalian MAPKKK activity in a yeast pheromone-induced MAPKpathway (K. Yamaguchi et al., Science, Vol. 270, p. 2008 (1995); K. Irieet al., Science, Vol. 265, p. 1716 (1994)). An activated form of TAK1(TAK1ΔN) can substitute for Ste11 MAPKKK activity.

Specifically, the pheromone-activated MAPK pathway consists of Ste11,Ste7, and Fus3 or Kss1 kinases, which correspond to MAPKKK, MAPKK andMAPK, respectively. These yeast protein kinases act sequentially totransduce signals to the transcription factor Ste12, upon which Ste12 inturn activates transcription of mating-specific genes such as FUS1 (I.Herskowitz, Cell, Vol. 80, p. 187 (1995); D. E. Levin et al., Curr.Opin. Cell Biol., Vol. 7, p. 197 (1995); J. Schultz et al., Jr. Curr.Opin. Gene Dev., No. 5, p. 31 (1995)).

The FUS1p::HIS3 reporter gene comprises the FUS1 upstream activatingsequence linked to the HIS3 open reading frame, and signal activity ofthe his3ΔFUS1p::HIS3 strain may be monitored by the ability of cells togrow on SC-His medium (His⁻ phenotype).

Strain his3Δste11ΔFUS1p::HIS3STE7^(P368) (proline substitution atserine-368) has a His⁻ phenotype (K. Irie et al., Science, Vol. 265, p.1716 (1994)).

Expression of TAK1ΔN in this strain confers a His⁺ phenotype (K.Yamaguchi et al., Science, Vol. 270, p. 2008 (1995)). Thus, theactivated form of TAK1 may substitute for Ste11 activity in anSte7^(P368)-dependent manner. However, expression of the full-lengthTAK1 does not complement the ste11Δ mutation, suggesting that the yeastdoes not have the putative activating factor for TAK1 (K. Yamaguchi etal., Science, Vol. 270, p. 2008 (1.995)).

The GAD-TAB1 constructs were tested for their ability to complement theste11Δ mutation in the presence of TAK1, using the yeast MAPK pathway.Specifically, yeast strain SY1984-P (his3Δste11ΔFUS1p::HIS3STE7^(P368))was transformed with pNV11-HU11 (TAK1ΔN)+pGAD10 (GAD) (Clontech),pNV11-HU11F (TAK1)+pGAD10, pNV11-HU11F+pGAD-TAB1 or pNV11+pGAD-TAB, andthe transformants were folded onto an SC-His plate and incubated at 30°C.

The aforementioned strain SY1984-P is SY1984 (his3Δste11ΔFUS1p::HIS3)transformed by plasmid pNC318-p368 containing STE7^(P368) under thecontrol of CYC1 promoter (K. Irie et al., Science, Vol. 265, p. 1716(1994)). The aforementioned plasmids pNV11-HU11 and pNV11-HU11Frespectively express the shortened TAK1ΔN (amino acids 21-579) and thefull-length TAK1-under the control of TDH3 promoter (K. Yamaguchi et al,Science, Vol. 270, p. 2008 (1995)).

The results are shown in FIG. 2. The left panel indicates whether theyeast strain tested expressed TAK1ΔN or TAK1, and whether GAD-TAB1 wassimultaneously expressed or not. The right panel shows the growth of thecells on the SC-His plate. Each of the patches represents the resultsfor an independent transformant.

The GAD-TAB1 and TAK1 simultaneous transformant restored the effect ofthe Ste11 deletion. This indicates that TAB1 reinforces the function ofTAK1.

Example 4

In order to determine whether TAK1 activity is increased inTAB1-expressing yeast, an expression DNA vector containing TAK1 carryingthe hemagglutinin (HA)-derived C-terminal epitope and a catalyticallyinactive TAK1 mutant [TAK1-K63W wherein lysine at position 63 of theATP-binding site is replaced with tryptophan (K. Yamaguchi et al.,Science, Vol. 270, p. 2008 (1995)] was used to transform yeast cells inthe absence and in the presence of the TAB1 gene.

The DNA sequence coding for an epitope recognized by the HA-specificmonoclonal antibody 12CA5 was joined in frame with the TAK1-codingsequence and TAK1-K63W C-terminus by the polymerase chain reaction(PCR). All of the constructs were expressed by TDH promoter. The TAB1expression plasmid pGAP-HTH9M expresses 68 C-terminal amino acids.YEpGAP112 is a multicopy plasmid TRP1 containing TDH3 promoter [H. Bannoet al., Mol. Cell Biol. 13, 475 (1993)].

A sequence coding for the 68 C-terminal amino acids of TAB1 wasamplified by the PCR using the 5′ primer: 5′-GAGAATTCATGCGGCAAAGC-3′(SEQ ID NO: 2) containing the EcoRI site and ATG codon and the3′-primer: 5′-GGGTCGACTACGGTGC-3′ (SEQ NO: 3) containing the SalI site.A 240 bp EcoRI-SalI fragment produced by PCR was inserted into theEcoRI-SalI gap of YEpGAP112 to construct pGAD-HTH9M.

The results are shown in FIG. 3. As described above, yeast strain SY1984was transformed with the aforementioned plasmid coding for TAK1-HA andplasmid coding for TAK1-K63W, and the empty vector YEpGAP112(−) orpGAP-HTH9M(+) coding for TAB1 was additionally inserted into thetransformant. TAK1-HA(−) or TAK1-K63W-HA(KN) was immunoprecipitated fromeach of the cell extracts and the immunoprecipitates were subjected toin vitro kinase assays. Specifically, 60 ml of yeast cell culture wasallowed to grow to an optical density of 0.8 at 600 nm, and a cellextract was prepared with a cytolytic buffer solution (K. Irie et al.,Science, Vol. 265, p. 1716 (1994)) and then separated by centrifugationat 100,000 g for 30 minutes.

The supernatant was subjected to immunoprecipitation with an antibodyagainst HA. That is, a portion (300 μl) of the supernatant was mixedwith 2 μl of antibody and 90 μl of Protein A-Sepharose, and theimmunocomplex was washed 3 times with a cytolytic buffer solution andused for the kinase assay (K. Yamaguchi et al., Science, Vol. 270, p.2008 (1995)). Immunoblot analysis of each immunoprecipitate with theHA-specific monoclonal antibody 12CA5 demonstrated that approximatelythe same amount of TAK1-HA or TAK1-K63W-HA was recovered in each sample.This suggests that expression of TAB1 does not affect the amount of TAK1expression.

The immunoprecipitated TAK1 was assayed based on the ability to activaterecombinant XMEK2 (SEK1), with the recombinant XMEK2 (SEK1) activitybeing assayed based on its ability to phosphorylate catalyticallyinactive (KN)p38 (MPK2) (K. Yamaguchi et al., Science, No. 270, p. 2008(1995)). After electrophoresis, phosphorylation of KN-p38 (MPK2) wasdetected by autoradiography. No extract exhibited a kinase assay valuewithout the enzyme extract. This level corresponds to the XMEK2 basalactivity. The experiment was conducted at least 3 times, giving the sameresults each time.

The results are shown in FIG. 3. The results of kinase assay for TAK1-HAand TAK1-K36W-TAK1 indicate that TAB1 increases TAK1 kinase activity.The activity increase was not observed for immunocomplexes from cellsexpressing TAK1-K63WKN and TAB1, indicating that the observed kinaseactivity was attributable to TAK1. These results demonstrate that TAB1activates TAK1 kinase activity by directly binding to the catalyticdomain of TAK1.

Example 5

To obtain the full-length coding sequence for TAB1, a human kidneylibrary was screened using as a probe the aforementioned partialsequence of TAB1 cDNA obtained from the yeast 2-hybrid system. Twoindependent clones carried 3.1 kb cDNA containing a single open readingframe (ORF) starting from the initial methionine codon matching theKozak consensus. The 5′-terminus was identified by the 5′ RACE methodusing 5′-RACE-Ready cDNA (Clontech).

The proposed N-terminal nucleotide sequence of the coding sequence(CCAAATGG) corresponds to the Kozak consensus (M. Kozak, J. Cell Biol.Vol. 108, p. 229 (1989)), and the ATG codon is not present before it.

The TAB1 nucleotide sequence was determined by the dideoxynucleotidechain termination method. An amino acid sequence was deduced from thenucleotide sequence of the full-length TAB1 cDNA. As a result, twodifferent clones were obtained with cytosine and adenine as the 185thnucleotide, respectively. The clone with cytosine as the 185thnucleotide encodes for serine as the 52nd amino acid, and the clone withadenine as the 185th nucleotide encodes arginine as the 52nd amino acid.

The nucleotide sequence of the clone with cytosine as the 185thnucleotide is shown in SEQ NO: 7, and its amino acid sequence is shownin FIG. 4 and in SEQ ID NO: 1. The nucleotide sequence of the clone withadenine as the 185th nucleotide is shown in SEQ ID NO: 6, and its aminoacid sequence is also shown in SEQ ID NO: 4.

The cDNA of the clone with cytosine as the 185th nucleotide wassubcloned at the EcoRI and SmaI sites of pBS to prepare plasmidTABI-f-4, while the cDNA of the clone with adenine as the 185thnucleotide was subcloned at the EcoRI site of pBS to prepare plasmidpBS-TAB1. E. coli containing plasmid pBS-TAB1 was named Escherichia coliHB101 (pBS-TAB1) and was deposited at the National Institute ofBioscience and Human Technology Agency of Industrial Science andTechnology on Apr. 19, 1996 as FERM BP-5508. E. coli containing plasmidTABI-f-4 was named Escherichia coli DH5α (TABI-f-4) and was deposited atthe National Institute of Bioscience and Human Technology Agency ofIndustrial Science and Technology on Jul. 19, 1996 as FERM BP-5599.

The following experiment was conducted using the clone having thenucleotide sequence shown in SEQ ID NO: 7.

In FIG. 4, A=Ala, C=Cys, D=Asp, E=Glu, F=Phe, G=Gly, H=His, I=Ile,K=Lys, L=Leu, M=Met, N=Asn, P=Pro, Q=Gln, R=Arg, S=Ser, T=Thr, V=Val,W=Trp and Y=Tyr. The 68 C-terminal amino acids of GAD-TAB1 isolatedusing the yeast 2-hybrid system are boxed.

The N-terminal sequence of TAK1 is aligned to show the region withsimilarity to the same segment of TAK1. The identical and conservedamino acids with respect to those of TAK1 are marked with asterisks anddots, respectively.

The ORF suggested a protein of 504 amino acids having a molecular sizeof 55 kDa, without clear similarity to any known protein and without anymotif indicating biological function.

Example 6

The expression patterns of TAB1 mRNA in different human cells wereanalyzed by Northern blotting. Human tissue blots (Clontech) of mRNAisolated from 16 tissues were probed with ³²P-labelled TAB1 cDNA, andsubjected to autoradiography. The results are shown in FIG. 5. Each ofthe lanes contained 2 μg of mRNA. The probe was labelled with[α-³²P]-dCTP using a Multiprime Labeling Kit (Amersham), and hybridizedas described by H. Shibuya et al.,. Nature, Vol. 357, p. 700 (1992). Amajor transcription product of about 3.5 kb was detected in all of thetissues tested.

Example 7

In order to confirm association of TAB1 and TAK1 in mammalian cells, anexpression vector producing HA epitope-labelled TAK1 (HA-TAK1) (K.Yamaguchi et al., Science, Vol. 270, p. 2008 (1995)) and an expressionvector producing Myc epitope-labelled TAB1 (Myc-TAB1) were used fortransient transfection of MC3T3-E1 murine osteoblasts (S. Ohta et al.,FEBS Lett., Vol. 314, p. 356 (1992)). The latter plasmid was obtained inthe following manner.

The full-length TAB1 cDNA was subcloned in pCS2MT vector containing 6copies of the Myc epitope (LEQKLISEEDLN (SEQ ID NO: 5)) (single letteramino acid sequence notation) recognized by the Myc-specific monoclonalantibody 9E10 (D. L. Tumer et al., Genes Dev., Vol. 8, p. 1434 (1994)).In the plasmid thus obtained, pCS2MT•TAB1, the Myc epitope tag is linkedin frame with the DNA sequence corresponding to the N-terminus of TAB1.pCSA2MT-TAB1 was digested with BamHI and XbaI. The fragment was isolatedand inserted at the EcoRI-XbaI site of the mammalian expression vectorpEF. This plasmid causes expression of TAB1 from the human elongationfactor 1α (EF1α) promoter.

The cell extract was subjected to immunoprecipitation with theHA-specific monoclonal antibody 12CA5 (lane 2 in FIG. 6), theMyc-specific monoclonal antibody 9E10 (lane 3 in FIG. 6) or a controlnonimmune IgG (lane 4 in FIG. 6). The immunocomplex was washed andseparated by SDS-PAGE, and then transferred to nitrocellulose forimmunoblotting using the Myc-specific antibody (top lanes of FIG. 6) andHA-specific antibody (bottom lanes of FIG. 6).

The cell extracts were then immediately subjected to immunoblot analysis(lane 1 of FIG. 6). As FIG. 6 shows, a considerable amount of Myc-TAB1was detected in each immunoprecipitation, indicating that TAK1 can beimmunoprecipitated with TAB1. A reciprocal experiment blotting theimmunoprecipitated protein with the HA-specific antibody confirmedassociation of TAB1 and TAK1. These experiments indicate that TAB1 canassociate with TAK1 in mammalian cells as in yeast.

Example 8

It was investigated whether overexpression of TAB1 can activate TAK1kinase activity. MC3T3-E1 cells were transiently transfected withHA-TAK1 in the presence of (+) and in the absence of (−) Myc-TAB1. Thecells were treated (+) or untreated (−) with 20 ng/ml TGF-β1 for 10minutes and then HA-TAK1 was immunoprecipitated in the manner describedin Example 3, after which the kinase activity was assayed. Specifically,a portion of the immunoprecipitate was immunoblotted with HA-specificantibody. The results are shown in FIG. 7.

The activity is given as a fold increase relative to the amount ofHA-TAK1 from unstimulated cells, and is expressed as mean±SEM from atleast 3 experiments (top graph in FIG. 7). HA-TAK1 did not directlyphosphorylate KH-p38(MPK2) (K. Yamaguchi et al., Science, Vol. 270, p.2008 (1995)). The middle panel is the autoradiogram showingphosphotylation of KN-p38(MPK2). The lower panel shows immunoblotanalysis of each of the immunoprecipitates with the HA-specificmonoclonal antibody 12CA5, where it is seen that approximately the sameamount of TAK-HA was recovered in each sample. The data shown in themiddle and lower panels are from typical experiments.

The in vitro assay of the TAK1 immunoprecipitation suggests that TAK1activity was stimulated in cells transfected with TAB1 even in theabsence of TGF-β. Activation of TAK1 by overexpression of TAB1 wascomparable to the activation observed in cells stimulated with TGF-βwhich expressed only HA-TAK1.

Example 9

TGF-β causes rapid increase in the amount of mRNA coding for plasminogenactivating factor inhibitor-1 (PAI-1) (M. R. Keeton et al., J. Biol.Chem., Vol. 266, p. 23048 (1991)). Overexpression of the activated formof TAK1 (TAK1ΔN) results in constitutive activation of a reporter genecontaining the luciferase gene under the control of the TGF-β-induciblePAI-1 gene promoter (K. Yamaguchi et al., Science, Vol. 270, p. 2,008(1995)). We investigated whether overexpression of TAB1 leads toactivation of the luciferase reporter gene.

MvlLu cells were transiently transfected by the calcium phosphate method(H. Shibuya et al., Nature, Vol. 357, p. 700 (1992)) using a reporterplasmid p800neoLUC (M. Abe et al., Analyt. Biochem., Vol. 216, p. 276(1994)) and the TAB1-expressing plasmid pEF-TAB1 or TAK1-encodingexpression plasmid (K. Yamaguchi et al., Science, Vol. 270, p. 2008(1995)). Plasmid pEF-TAB1 contains the full-length TAB1 coding sequenceunder the control of EF1α promoter, and was constructed by cleaving pEFwith EcoRI and inserting the EcoRI fragment from plasmid TABI-f-4.

The plasmid TABI-f-4 was constructed by subcloning TAB1 cDNA at theEcoRI and SmaI sites of pBS. The cells were incubated for 20 hours withand without 30 ng/ml of human TGF-β1, an extract was prepared; and theluciferase was assayed (H. Shibuya et al., Mol. Cell Biol., Vol. 14, p.5812 (1994)). The luciferase activity was compensated based onexpression of β-galactosidase.

Specifically, the transfection efficiency was compensated bysimultaneous transfection with pXeX-β-Gal vector (A. D. Johnson et al.,Gene, Vol. 147, p. 223 (1994)) in all of the luciferase reporterexperiments. Measurement of β-galactosidase was made according to theinstructions of the manufacturer (Clontech), using the cell lysateprepared for the luciferase measurement. The luciferase activity wasgiven as the fold-increase with respect to the activity of unstimulatedcells transfected with the vector. All of the transfection andluciferase measurements were conducted at least 5 times, withtriplicates of each experiment.

The results are shown in FIG. 8. Here, KN indicates the catalyticallyinactive TAK1-K63W. The data is expressed as the mean±SEM of theluciferase activity from triplicates in a representative experimentoverexpression of both TAK1 and TAB1 induced expression of the reportergene even in the absence of TGF-β, but overexpression of only TAK1 orTAB1 had virtually no effect on the constitutive amount of luciferaseactivity. These experimental results indicate that TAB1 reinforces TAK1activity in mammalian cells.

Although overexpression of the TAK1-K63W mutant inhibitedTGF-β-stimulated luciferase activity (K. Yamaguchi et al., Science, Vol.270, p. 2008 (1995)), this is presumably due to sequestering ofessential elements in the pathway. On the other hand, overexpression ofTAB1 reduces the inhibiting effect of TAK1-K63W, suggesting thepossibility that TAB1 is absorbed by overexpression of TAK1-K63W.

Example 10

The 68 C-terminal amino acids of TAB1 [TAB1 (437-5.04)] were sufficientto bind to and activate TAK1, suggesting that the N-terminal domain ofTAB1 performs a regulatory role on the function of TAB1. To test thispossibility, a shortened form of TAK1 lacking the C-terminalTAK1-binding domain (TAB1 (1-418)) was constructed. MvlLu cells weretransiently transfected with p800nedUC reporter and an expression vectorcoding for TAB8 (1-418) or TAB1 (full-length) in the amounts shown inFIG. 9, and these were complemented with the pEF control vector.

The expression vector coding for TAB1 (1-418) was constructed in thefollowing manner. The 1.3 kb EcoRI-HincII fragment of plasmid TABI-f-4(containing the TAB1 N-terminal region of amino acids 1-418) wassubcloned in pKT10 vector to construct pKT10-TAB1 (1-418). pEF wascleaved with EcoRI and SalI, and the EcoRI-SalI fragment from pKS10-TAB1(1-418) was inserted therein to construct pEF-TAB1 (1-418).

Next, the cells were incubated for 20 hours with and without 30 ng/ml ofhuman TGF-β1, and the cell lysate was measured for luciferase activity.The values were expressed as fold induction in terms of a percent withrespect to the control cells transfected with pEF. No induction ofluciferase with TGF-β (1-fold induction) corresponds to 0%. All of thetransfection and luciferase measurements were conducted at least 3times, and a series of 3 of each of the experiments were conducted. Thedata is expressed as the mean±SEM of the luciferase activities fromtriplicates in a representative experiment.

The results are shown in FIG. 9. Overexpression of TAB1 (1-418) in MvlLucells suppressed activity of the reporter gene induced by TGF-βstimulation. Thus, TAB1 (1-418) acts as the dominant negative inhibitoron gene expression induced by TGF-β. These results indicate that TAB1plays a role in TGF-β signaling.

The mechanism of induction of TAK1 activation by TAK1 believed to bethat TAB1 binding to TAK1 induces the necessary conformational changesfor activation. Since removal of the 20 N-terminal amino acids of TAK1causes constitutive activation of the protein kinase, this suggests thatthe N-terminal domain hinders the catalytic domain, thus inhibitingkinase activity (K. Yamaguchi et al., Science, Vol. 270, p. 2008(1995)). TABS may eliminate the negative control domain of TAK1 from itscatalytic domain. The C-terminus of TAB1 which functions as theTAK1-binding site contains a serine- and threonine-rich region similarto the region found at the N-terminus of TAK1. Therefore, TAB1 isprobably an important signaling intermediate between TGF-β and TAK1MAPKKK.

1. A method for producing a TAB1 protein or a fragment thereof,comprising: (a) culturing a host that is transformed with a DNA encodingsaid TAB1 protein or a fragment thereof in a culture medium, whereinsaid host is a mammalian cell or yeast cell; and (b) isolating the TAB1protein or fragment thereof from the culture medium wherein said TAB1protein comprises the amino acid sequence of SEQ ID NO: 1; and whereinsaid fragment of TAB1 protein comprises the amino acid sequence from theGin residue at position 437 to the Pro residue at position 504 of SEQ IDNO: 1.